Nanotechnology: Toxicological considerations. Terry Tetley · Effects of ambient environmental...
Transcript of Nanotechnology: Toxicological considerations. Terry Tetley · Effects of ambient environmental...
Nanotechnology: Toxicological considerations.
Terry Tetley
Lung Cell Biology
Section of Pharmacology and Toxicology National Heart and Lung Institute
Imperial College
AIR POLLUTION
Friday 5 to Tuesday 9 December 1952
Estimated that up until 8 December 4,000 people had died prematurely
and 100,000 more were made ill.
Recent research suggests that the total number of fatalities was considerably
greater, at about 12,000
Effects of ambient environmental particulate air pollution on cardio vascular health
• Increases in PM2.5 from a few hours up to weeks causes increased ischemic heart disease events and hospitalisation
• Increased myocardial infarct, both acute and chronic exposure, and increased risk of death from myocardial infarct with chronic exposure
• Raised blood pressure
• Reduced heart rate variability
• Development (in previously healthy individuals) and acceleration of atherosclerosis
• Deaths form heart attacks
• Deaths from strokes
Effects of ambient environmental particulate air pollution on respiratory health.
• Increased coughing and wheezing, particularly children
• Increased use of medication for asthma
• Attacks of asthma in patients with pre-existing asthma, especially children
• Exacerbation of chronic obstructive pulmonary disease (COPD) and associated increased hospital admissions (doubling for each 10mg/m3 increase in PM2.5) and increased rate of decline in FEV1
• Reduced growth in lung function in children
• Increased risk of microbial infection
• Deaths from cancer and other respiratory causes
• Prenatal exposure relates to reduced lung function/function and increased airway hyperresponsiveness, airway inflammation, mucus secretion
LONDON, DECEMBER 1952
AIR POLLUTION: 1952 SMOGS – MIX OF SOOT AND FOG
Today: Deisel exhaust particles are <100nm in diameter
ENGINEERED NANOPARTICLES?
EFFECTS UNKNOWN
Nanoparticles/nanomaterials are structures which have at least one dimension of 100nm or less. Include nanofibres, nanotubes as well as spherical structures with a diameter of <100nm.
Nanomaterials
Why use nanoparticles?
• When materials enter the nano-range, their properties alter from that of their micron-sized equivalents.
• Nanoparticles have enhanced thermal, magnetic, electrical and optical properties
• As size decreases, the surface area:volume ratio increases
• The surface is more reactive, improving drug labelling
Nanoparticle: Less than 100nm in one dimension
1 1cm3 sugar cube,
1 x 1015 100nm3 cubes
6.4 x 1016 25nm3 cubes
Nanoparticles can be natural, anthropogenic or manufactured
Sugar cube analogy
Nanoparticle use, number of products listed as containing nanoparticulate material
Over 1300 products-
Medicine Engineering Construction industry Cosmetics Clothing Food Sports goods
Antibacterial sprays and medical products
Cosmetics, sun cream
Sports goods
Low calorie food products
Diagnosis, treatment
Clothing
Use of engineered nanoparticles
Nanoparticle use
2002 – Publication of Prey, a novel by Michael Crichton. A medic with a vivid imagination. The book describes how an artificial swarm of nanorobots develop intelligence and threaten their human inventors. The novel generated concern within the nanotechnology community that the public perception of nanotechnology might be affected, creating fear of a similar scenario in real life.
Nanotoxicology Following concerns raised by Prince Charles and others, the Royal Society and Royal Academy of Engineering published a report, Nanoscience and nanotechnologies: opportunities and uncertainties, July 2004. Nanotoxicology emerged as a subject in its own right. http://www.nanotec.org.uk/finalReport.htm.
Perceived risks: • Occupational exposure
• Exposure during use/application/disposal
• Accidental exposure
Concerns: • Safety: Potential adverse effects
via inhalation- lungs, dermal-skin, ingestion-gut, systemic delivery
(e.g. intravenous administration)
• Susceptible subpopulations
• Implications for society
Risks and concerns
Deposition and impact of inhaled nano-sized ambient air pollution particles
Structure of the lung
TT1 cells - Transformed human alveolar epithelial type-1-like cell (TT1) model. Kemp, S. J. et al Am. J. Respir. Cell Mol. Biol. 2008
AT2 cells - Primary alveolar epithelial type 2 cells, isolated from normal regions of human lung tissue.
In vitro alveolar models
AM – Alveolar macrophages lavaged from human lung tissue
Human lung cell monolayer
Alveolar epithelium, alveolar macrophages, fibroblasts,
Incubated with nanoparticles
Up to 24 hours (acute)
Medium Cells
Pro-inflammatory mediators Particle uptake, Viability, Cytokines, chemokines Reactive oxygen species
Nanoparticle exposure models
4hr
Carboxyl
24hr
Neutral
ATII cell uptake of 50nm polystyrene latex nanoparticles
4hr
Carboxyl
24hr
Neutral
ATI cell uptake of 50nm polystyrene latex nanoparticles
0 30 60 90 120 150 180 210 2400
1000
2000
3000
4000
5000
6000
7000
Time (mins)
MFI
0 30 60 90 120 150 180 210 2400
1000
2000
3000
4000
5000
6000
7000
Time (mins)
MFI
50nm 100nm
ATI cell uptake of 50nm and 100nm polystyrene latex nanoparticles
Amine-modified Carboxyl-modified Unmodified
Particle cytotoxicity
0 50 100 150 2000
20
40
60
80
100UnmodifiedAmine ModifiedCarboxyl Modified
Dose (g/ml)
% V
iabi
lity
0 50 100 150 2000
20
40
60
80
100UnmodifiedAmine ModifiedCarboxyl Modified
Dose (g/ml)
% V
iabi
lity
50nm Nanoparticles
0 50 100 150 2000
20
40
60
80
100
Dose (g/ml)
UnmodifiedAmine ModifiedCarboxyl Modified
% V
iabi
lity
100nm Nanoparticles
Carboxyl-modified NP
A
E
Amine-modified NP
Hopping probe ion conductance microscopy of AT1 epithelial cells exposed to carboxyl-modified and amine-modified 50nm
latex particles for 4 hours.
Carboxyl-modified NP
A
E
Amine-modified NP
Hopping probe ion conductance microscopy of AT1 epithelial cells exposed to carboxyl-modified and amine-modified 50nm
latex particles for 4 hours.
Ruenraroengsak et al. Respiratory epithelial cell cytotoxicity and membrane damage (holes) caused by amine-modified nanoparticles, Nanotoxicology 2012, 6:94-108
500nm
Cellular fate of nanoparticles
Engineered nanoparticles/nanomaterials • Carbon-based – fullerenes, carbon nanotubes, wires, graphene etc
• Metals – gold, silver
• Metal oxides – titanium dioxide (self cleaning surfaces), zinc oxide (both in
sunscreens), copper oxide, cerium oxide (in deisel to reduce particle numbers)
• Organic/soft – as synthesised for drug delivery
• Quantum dots <10nm diameter
ARGYRIA – was he born with a silver spoon in his mouth?
Medical applications of silver nanoparticles
Au 5 nm
Au 15 nm
1 μg/ml 10 μg/ml 25 μg/ml 50 μg/ml
Differences in cellular uptake of gold and silver nanoparticles
Phagocytic alveolar macrophage cell exposure (4 hr)
Does this impair phagocytic activity? eg during bacterial infection
Au 5 nm
1 μg/ml 10 μg/ml 25 μg/ml 50 μg/ml
Au15 nm
Alveolar epithelial Type 1 cell exposure (4 hr)
Differences in cellular uptake of gold and silver nanoparticles
Au 5 nm
Au 15 nm
Alveolar epithelial type 2 cell exposure (4 hr)
1 μg/ml 10 μg/ml 25 μg/ml 50 μg/ml
Differences in cellular uptake of gold and silver nanoparticles
Effect of Ag on ATI cells – not cytotoxic, but stimulate pro-inflammatory mediators
Ag (g/ml)
% C
ell V
iabi
lity
0 10 20 30 40 500
20
40
60
80
100 10nm Ag Spheres50nm Ag SpheresAg Nanowires
Ag (g/ml)IL
-8 (p
g/m
l)
0 10 20 30 40 500
50100150200250300350400
10nm Ag Spheres50nm Ag SpheresAg NanowiresPristine
Ag (g/ml)
% C
ell V
iabi
lity
0 10 20 30 40 500
20
40
60
80
100 10nm Ag Spheres50nm Ag SpheresAg Nanowires
Silver spray products NAME DESCRIPTION CODE
Colloidal Silver Spray Used as cosmetic spray, dermatologic applicatio S1CS
Schudheo Used to prevent unpleasant odors in shoes S3CS
Skin and Body Protectant Used for skin enhancement S5CS
General Hygiene Antiseptic Used to clean skin S6CS
Mesosilver Dermal application as antifungal spray. S7HS
Colloidal Silver Nasal Spray Used to improve breathing S8HS
Sovereign Silver Used as dietary supplement S9HS
Smelly Shoe Spray Used to prevent unpleasant odors in shoes S10OS
Silver Care Used as cosmetic for skin protection S11CS
Ultra Colloidal Silver Spray Used orally as a dietary supplement S12HS
Nanofix Used to clean surfaces S13OS
Nan Health Silver Used orally to prevent diseases (no built-in sprayer)
S2HO
Silver 22 Used orally as a dietary supplement (no built-in sprayer)
S4HO
Slide: courtesy Gedi Mainelis, Rutgers
Smelly Shoe Spray
• 3.86 µg/ml Colloidal Silver
• Isopropanol
• Water
• Peppermint essential oil
MesoSilver Nanofix Silver Nasal Spray
• 15.86µg/ml Colloidal silver
• Deionized water
• 1.21µg/ml silver
• 11.096 µg/ml silver
• Deionized water
SILVER CONTAINING PRODUCTS
S1 S2
• 505.4ug/ml Pyrithione Zinc
• SDA-40 Alcohol
• Isopropyl Myristate
• SLS needles • Undecy lenic acid
• 5115µg/ml Zinc
• Vegitable Glycerin
• Peppermint Oil
• Clovebud oil
• Echinacea extract
• Menthol
DermaZinc Therazinc
Zinc containing Products
Z1 Z2
Effect of silver products on type-1 epithelial cell viability 24 hr exposure
0.048 0.096 0.3877 0.198 0.397 1.5882
particle concentration micrograms/ml
6.32 12.65 50.6 63.93 127.85 511.5
Effect of zinc products on Type -1 epithelial cell viability 24 hour exposure
particle concentration micrograms/ml
Nanoparticles can induce reactive oxygen species in alveolar cells
Control
AT2
+NPs NPs +NAC +NPs NPs+NAC +NPs NPs+NAC
MAC
i j k l
m n o p
TT1 a b c d
e f g h
T=4h
q r s t
u v w x
Co-application of N-acetylcysteine prevents induction of ROS in epithelial cells, but has no effect on macrophages
DOSE
Silver-induced genotoxicity
nt 1 10 30 500
2
4
6
8
10 ***
**
C (g/ml)
H2A
.X /
GA
PD
HGAPDH
H2A.X
nt 1 10 30 50 Ag (μg/ml) Induction of double-stranded DNA breaks
Collaborative in vivo studies show that inhalation of silver causes acute: • Inflammation
• Reduction in elastance/elastic
recoil
• Reduced lung surfactant function
"
Qualities of graphine:
•A thin flake of carbon, one atom thick
•Thinnest, strongest material ever
•Conducts electricity as effectively as copper
•Conducts heat better than all other materials
•Almost completely transparent, yet so dense,
not even helium can pass through it.
Human hair 70m
Nanowire 1/10,000th
Carbon nanotubes
Multi-walled and single walled
Less than 100nm diameter.
Can reach many micrometres in length.
Use of carbon nanotubes, wires, fibres: Engineering Electronics Construction industry Cosmetics Clothing Sports goods Medicine
Bhirde,A.A. et al. ACS Nano 3, 307-316 (2009).
Bhirde,A.A. et al. ACS Nano 3, 307-316 (2009).
-EGF
+EGF
Carbon nanostructures may exist in many formats including tubes, wires and fibres
Crocidolyte asbestos Chrysotile asbestos
Inhalation of asbestos can cause lung disease
NORMAL LUNG
ASBESTOSIS
LUNG CANCER & MESOTHELIOMA
TT1 - 200nm 4VP CNTs, T=24h
Transmission EM of TT1 cell uptake of 200nm MWCNTs
TT1 - 700nm 4VP CNTs, T=24h
28.4 1
7.4
21.9
20.0
20.2
SHORT MWNTs LONG MWNTs
Effect of long and short multiwalled carbon nanotubes on human alveolar cells
AT2 cells
0 0.1 1 5 10 50 1000
102030405060708090
100CNT ACNT BCNT CCNT DCNT E
**
* ***
CNT sample (g/ml)
Via
bilit
y (%
of
Con
trol
)
AT2 cells
0 0.1 1 5 10 50 1000
102030405060708090
100CNT ACNT BCNT CCNT DCNT E
**
* ***
CNT sample (g/ml)
Viab
ility
(% o
f Con
trol) 15-20m
Long) 3-5m (medium) 0.6-2m (short)
TT1 cells
0 0.1 1 5 10 25 50 100 5000
102030405060708090
100110
CNT ACNT BCNT CCNT DCNT E
***
*
******
***
******
********
CNT sample (g/ml)
Viab
ility
(% o
f Con
trol
)
Effect of long and short MWCNT on cell viability (MTT) With and without Curosurf (pig surfactant)
AM cells
0 0.1 1 5 10 50 1000
102030405060708090
100CNT ACNT BCNT CCNT DCNT E
***
******
* *
CNT sample (g/ml)
Via
bilit
y (%
of
Con
trol
)
long
short
IL-8 release from TT1, AT2 and AMs exposed to short and long MWCNTs
TT1 cells
0
500
1000
1500
200000.111050
***
***
** * *
Purity
Length 20m 3m0.6m 20m
48% 74% 97% 97%
CNT sample (g/ml)
IL-8
(pg/
ml)
TT1 cells
0
500
1000
1500
200000.111050
***
***
** * *
Purity
Length 20m 3m0.6m 20m
48% 74% 97% 97%
CNT sample (g/ml)
IL-8
(pg/
ml)
AT2 cells
0
2000
4000
6000
8000
10000
1200000.111050
******
*
******
*****
Purity
Length 20m 3m0.6m 20m
48% 74% 97% 97%
CNT sample (g/ml)
IL -
8 (p
g/m
l)AT2 cells
0
2000
4000
6000
8000
10000
1200000.111050
******
*
******
*****
Purity
Length 20m 3m0.6m 20m
48% 74% 97% 97%
CNT sample (g/ml)
IL -
8 (p
g/m
l)
TT1 cells AT2 cells TT1 cells
0
500
1000
1500
200000.111050
***
***
** * *
Purity
Length 20m 3m0.6m 20m
48% 74% 97% 97%
CNT sample (g/ml)
IL-8
(pg/
ml)
AMs
0
2000
4000
6000
8000
1000000.11
5010
***
***
* ****
Purity
Length 20m3m0.6m
97%
CNT sample (g/ml)
IL -
8 (p
g/m
l)
AMs
0
2000
4000
6000
8000
1000000.11
5010
***
***
* ****
Purity
Length 20m3m0.6m
97%
CNT sample (g/ml)
IL -
8 (
pg
/ml)
AMs
TT1 cells
0
500
1000
1500
200000.111050
***
***
** * *
Purity
Length 20m 3m0.6m 20m
48% 74% 97% 97%
CNT sample (g/ml)
IL-8
(pg/
ml)
AT2 cells
0
2000
4000
6000
8000
10000
1200000.111050
******
*
******
*****
Purity
Length 20m 3m0.6m 20m
48% 74% 97% 97%
CNT sample (g/ml)
IL -
8 (p
g/m
l)
AMs
0
2000
4000
6000
8000
1000000.11
5010
***
***
* ****
Purity
Length 20m3m0.6m
97%
CNT sample (g/ml)
IL -
8 (pg
/ml)
g/ml
• Shorter CNTs are more reactive with epithelial cells. • Longer CNTs are more reactive with macrophages.
Same pattern of response for IL-6 and MCP1 release
Frustrated phagocytosis is a common feature of mesothelioma
Frustrated phagocytosis of carbon nanotubes by human alveolar macrophages
Effect of MWCNT of different lengths on human lung fibroblast viability (n=4 subjects)
Effect of MWCNTs of different lengths on soluble collagen release by human lung fibroblasts
N=4
Development of granuloma and mesothelioma following injection of MWCNT into peritoneal cavity
Can Carbon Nanotubes cause fibrosis?
Ma-Hock,L. et al. Toxicol. Sci. 112, 468-481 (2009) Shvedova, A.A et al. Am J Physiol Lung Cell Mol Physiol. 2008 Oct;295(4):L552-65
Ag PVP
20nm 110nm
Curosurf Curosurf
g/ml Ag
IL-6 pro-inflammatory cytokine release by AT1 cells exposed to silver nanoparticles
67
SP-A
SP-D
Binding of surfactant proteins A and D to MWCNTs
36 72 108 KDa
KDa 43
SPA binding
SPD binding
MWCNTs Long (>15m) Short (<3m)
NT 1 5 10 25 50
AR MWCNT (µg/ml)
β-tubulin
SpD
SpA
Intracellular AT2 SP-A and SP-D following exposure to pristine Arkema MWNTs
Mercer et al, Particle and Fibre Toxicology, 2013 epub ahead of print
Verdict Some nanosized materials may have adverse health effects They need to be tested, safety procedures need to be put
into place
Imperial College London
MRC-HPA Centre for Environment and Health
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